The long term goal of this proposal is to establish the molecular framework that underlies Microtubule Organizing Centers (MTOCs). These are complex macromolecular assemblies that play essential roles in a wide variety of cellular functions including chromosome segregation, cytokinesis, fertilization, cell motility and intracellular trafficking. Given their critical role in cell division, it is not surprising that defects in the MTOCs assembly and function have important implications in disease, particularly in cancer and birth defects. The focus of this proposal is the spindle pole body (SPB) of the budding yeast Saccharomyces cerevisiae, which has long served as the paradigm for understanding MTOCs. The aim is to provide a high resolution picture of how this intricate multilayer structure assembles and is regulated. This will be accomplished by determining the three-dimensional structures of the SPB components and their macromolecular assemblies. The biological implications of these structures will be tested in vivo with an emphasis on the role of phosphoryation in SPB assembly. To this end, we have developed a strategy for expressing proteins of the SPB that contain coiled coils. The three dimensional structure of one component of the intermediate layer 2 has been determined and crystals for a second component have been obtained. This establishes the foundation for investigating the structure and function of the protein complexes that constitute the SPB. The specific aims for this proposal are: 1) to establish the underlying architecture of the intermediate layer 2 (IL2). This is a structure that forms a two-dimensional crystalline array which defines the lateral organization of the entire SPB. 2) to understand the organization of the central plaque, which is embedded in the nuclear envelope and establishes continuity between the nuclear and cytoplasmic sides of the SPB. 3) to define the molecular framework within the intermediate layer 1 which bridges the IL2 and the outer plaque. 4) to utilize the structural information as the basis for investigating the in vivo assembly and regulation of the SPB. This study falls into the high-risk high-payoff category of research proposals. It is high risk because it focuses on an enormous macromolecular assembly that is formed by a large number of unique protein-protein interactions. At the same time, the proposal is high payoff because little is known about the structure of this group of proteins. Thus any progress on these proteins will significantly advance our understanding of MTOCs. The strategies developed here to study the architecture of the SPB should be generally useful in facilitating the studies of other large macromolecular assemblies such as the centrosomes and cilia. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to establish a three dimensional model for the Microtubule Organizing Centers. These organelles play essential roles in cell division in all eukaryotes and have important implications in cancer and birth defects. The fundamental knowledge gained here will be of great value in understanding the molecular basis of disease.